Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation简

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.     

Enteric glia mediate neuronal outgrowth through release of neurotrophic factors

Previous studies have shown that transplanted enteric glia enhance axonal regeneration, reduce tissue damage, and promote functional recovery following spinal cord injury. However, the mechanisms by which enteric glia mediate these beneficial effects are unknown. Neurotrophic factors can promote neuronal differentiation, survival and neurite extension. We hypothesized that enteric glia may exert their protective effects against spinal cord injury partially through the secretion of neurotrophic factors. In the present study, we demonstrated that primary enteric glia cells release nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor over time with their concentrations reaching approximately 250, 100 and 50 pg/mL of culture medium respectively after 48 hours. The biological relevance of this secretion was assessed by incubating dissociated dorsal root ganglion neuronal cultures in enteric glia-conditioned medium with and/or without neutralizing antibodies to each of these proteins and evaluating the differences in neurite growth. We discovered that conditioned medium enhances neurite outgrowth in dorsal root ganglion neurons. Even though there was no detectable amount of neurotrophin-3 secretion using ELISA analysis, the neurite outgrowth effect can be attenuated by the antibody-mediated neutralization of each of the aforementioned neurotrophic factors. Therefore, enteric glia secrete nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-3 into their surrounding environment in concentrations that can cause a biological effect.     

Intraspinal transplantation of motoneuron-like cell combined with delivery of polymer-based glial cell line-derived neurotrophic factor for repair of spinal cord contusion injury

To evaluate the effects of glial cell line-derived neurotrophic factor transplantation combined with adipose-derived stem cells-transdifferentiated motoneuron delivery on spinal cord con-tusion injury, we developed rat models of spinal cord contusion injury, 7 days later, injected adipose-derived stem cells-transdifferentiated motoneurons into the epicenter, rostral and caudal regions of the impact site and simultaneously transplanted glial cell line-derived neuro-trophic factor-gelfoam complex into the myelin sheath. Motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery reduced cavity formations and increased cell density in the transplantation site. The combined therapy exhibited superior promoting effects on recovery of motor function to transplantation of glial cell line-derived neurotrophic factor, adipose-derived stem cells or motoneurons alone. These ifndings suggest that motoneuron-like cell transplantation combined with glial cell line-derived neurotrophic factor delivery holds a great promise for repair of spinal cord injury.     

In Vitro Enfolding of Olfactory Neurites by p75 NGF Receptor Positive Ensheathing Cells from Adult Rat Olfactory Bulb

Secondary cultures of adult rat olfactory bulb (OB) contained three different types of cell: (i) process-bearing cells; (ii) macrophage-like cells and (iii) fusiform cells. The immunohistochemical properties of process-bearing cells closely corresponded to those described for ensheathing glia in vivo. The most distinctive feature of these cells was their immunoreactivity for low affinity nerve growth factor receptor (NGFR). Process-bearing cells also shared the ultrastructural properties of ensheathing glia in vivo , as well as the ability to ensheath olfactory axons. In contrast, macrophage-like cells had the immunostaining properties of microglia, and fusiform cells were likely capillary endothelial cells.
Neurites outgrowing from olfactory epithelium explants, when co-cultured with adult OB cells, grew preferentially over NGFR positive cells. Olfactory neurites exhibited NGFR immunoreactivity and were enfolded by NGFR positive cells. After ensheathment, this immunoreactivity decreased from the neurite and disappeared from the glial membrane in contact with the neurite. However, NGFR immunoreactivity was maintained in the portion of the glial membrane not involved in ensheathing. In summary, ensheathing cells in vitro retained both the ultrastructure shown in vivo and the ability to ensheath olfactory neurites. The Schwann cell-like properties of ensheathing glia, could partially explain the permissibility of adult OB to axonal growth.     

Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors

Several groups have suggested that transplantation of marrow stromal cells (MSCs) promotes functional recovery in animal models of brain trauma. Recent studies indicate that tissue replacement by this method may not be the main source of therapeutic benefit, as transplanted MSCs have only limited ability to replace injured central nervous system (CNS) tissue. To gain insight into the mechanisms responsible for such effects, we systematically investigated the therapeutic potential of MSCs for treatment of brain injury. Using in vitro studies, we detected the synthesis of various growth factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3). Enzyme-linked immunosorbent assay (ELISA) demonstrated that MSCs cultured in Dulbecco's modified Eagle medium (DMEM) produced substantial amounts of NGF for at least 7 weeks, whereas the levels of BDNF, GDNF and NT-3 remained unchanged. In studies in mice, after intraventricular injection of MSCs, NGF levels were increased significantly in cerebrospinal fluid by ELISA, confirming our cell culture results. Further studies showed that treatment of traumatic brain injury with MSCs could attenuate the loss of cholinergic neuronal immunostaining in the medial septum of mice. These studies demonstrate for the first time that by increasing the brain concentration of NGF, intraventricularly transplanted MSCs might play an important role in the treatment of traumatic brain injury.     

Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose

Several studies have demonstrated the potential of olfactory ensheathing cells for the repair of central and peripheral nerve injury. However, the majority of these studies have been performed with olfactory ensheathing cells derived from the olfactory bulbs, situated inside the skull. A more clinically relevant source of olfactory ensheathing cells is the olfactory mucosa, located in the nose. To be successful, an autologous transplant of nasal ensheathing glia would require a large number of purified cells. To address this issue, we have focused our research on three neurotrophic factors, namely nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3). We show here that their respective receptors, TrkA, TrkB, TrkC, as well as p75(NTR) (the low affinity NGF receptor), are expressed in vitro by the nasal ensheathing cells; the three neurotrophins promote purification and proliferation of these glial cells, with an optimal concentration of 50 ng/ml; and human ensheathing cells can be easily biopsied and highly purified using a serum-free medium supplemented with NT3. This technique opens the door for clinical trials in which nasal ensheathing cells will be autotransplanted in humans suffering from nerve injury.     

Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague-Dawley rats weighing 250-300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.     

Glial cell line-derived neurotrophic factor modulates kindling and activation-induced sprouting in hippocampus of adult rats

Kindling, a phenomenon in which repeated electrical stimulation of certain forebrain structures leads to an increase in the evoked epileptogenic response, is widely used to investigate the mechanisms of epilepsy. Kindling also results in sprouting of the dentate gyrus mossy fiber pathway and triggers astrocyte hypertrophy and increased volume of the hilus of the dentate gyrus. Our previous studies showed that infusion of the neurotrophin nerve growth factor accelerated the behavioral progression of amygdala kindling and affected kindling-induced structural changes in the brain, whereas intrahilar infusion of another neurotrophin, brain-derived neurotrophic factor, delayed amygdala kindling-induced seizure development and reduced the growth in afterdischarge duration, but had little effect on kindling-induced structural changes. In this paper, we report the effects of infusion of glial cell line-derived neurotrophic factor, a neurotrophic factor of the TGF-beta superfamily having similar central nervous system neuronal targets as brain-derived neurotrophic factor. We show that continuous intraventricular infusion of glial cell line-derived neurotrophic factor inhibits the behavioral progression of perforant path kindling-induced seizures without affecting afterdischarge duration. In addition, we demonstrate that intraventricular administration of glial cell line-derived neurotrophic factor prevents kindling-induced increases in hilar area and blocks mossy fiber sprouting in the CA3 region of the hippocampus. Glial cell line-derived neurotrophic factor did not have a statistically significant effect on the mossy fiber density in the inner molecular layer. Our results raise the possibility that glial cell line-derived neurotrophic factor plays a role in kindling and activation-induced neural growth via mechanisms distinct from those of the neurotrophins.     

Human umbilical cord blood stem cells and brain-derived neurotrophic factor for optic nerve injury:a biomechanical evaluation

Treatment for optic nerve injury by brain-derived neurotrophic factor or the transplantation of human umbilical cord blood stem cells has gained progress, but analysis by biomechanical indicators is rare. Rabbit models of optic nerve injury were established by a clamp. At 7 days after injury, the vitreous body received a one-time injection of 50 μg brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood stem cells. After 30 days, the maximum load, maximum stress, maximum strain, elastic limit load, elastic limit stress, and elastic limit strain had clearly improved in rabbit models of optical nerve injury after treatment with brain-derived neurotrophic factor or human umbilical cord blood stem cells. The damage to the ultrastructure of the optic nerve had also been reduced. These findings suggest that human umbilical cord blood stem cells and brain-derived neurotrophic factor effectively repair the injured optical nerve, improve biomechanical properties, and contribute to the recovery after injury.     

Brain-derived neurotrophic factor/glial cell line-derived neurotrophic factor survival effects on auditory neurons are not limited by dexamethasone

Cochlear implant performance depends on the number of surviving excitable auditory neurons and prevention of degradation of nerve-electrode interaction caused by adverse tissue reactions. Glucocorticoids and neurotrophic factors are promising options for a possible therapeutic intervention. Neurons dissociated from the spiral ganglion of rats (3-5 days old) were cultivated with addition of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and the corticosteroid dexamethasone in various concentrations (25, 50, 100 ng/ml) and in combination with each other (100 ng/ml). The results suggest that a combination of brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor does not enhance spiral ganglion cell survival significantly when compared with single brain-derived neurotrophic factor treatment (100 ng/ml). In addition, dexamethasone application did not interfere with the survival-promoting effects of brain-derived neurotrophic factor or glial cell line-derived neurotrophic factor.     

Peripheral glial cell differentiation from neurospheres derived from adipose mesenchymal stem cells

Mesenchymal stem cells derived from bone marrow and adipose tissue are being considered for use in neural repair because they can differentiate after appropriate induction in culture into neurons and glia. The question we asked was if neurospheres could be harvested from adipose-derived stem cells and if they then could differentiate in culture to peripheral glial-like cells. Here, we demonstrate that adipose-derived mesenchymal stem cells can form nestin-positive non-adherent neurosphere cellular aggregates when cultured with basic fibroblast growth factor and epidermal growth factor. Dissociation of these neurospheres and removal of mitogens results in expression of the characteristic Schwann cell markers S100 and p75 nerve growth factor receptor and GFAP. The simultaneous expression of these glia markers are characteristic features of Schwann cells and olfactory ensheathing cells which have unique properties regarding remyelination and enhancement of axonal regeneration. When co-cultured with dorsal root ganglion neurons, the peripheral glial-like cells derived from adipose mesenchymal stem cells aligned with neuritis and stimulated neuritic outgrowth. These results indicate that neurospheres can be generated from adipose-derived mesenchymal stem cells, and upon mitogen withdrawal can differentiate into peripheral glial cells with neurotrophic effects.     

Extracellular matrix from human umbilical cord-derived mesenchymal stem cells as a scaffold for peripheral nerve regeneration

The extracellular matrix, which includes collagens, laminin, or fibronectin, plays an important role in peripheral nerve regeneration. Recently, a Schwann cell-derived extracellular matrix with classical biomaterial was used to mimic the neural niche. However, extensive clinical use of Schwann cells remains limited because of the limited origin, loss of an autologous nerve, and extended in vitro culture times. In the present study, human umbilical cord-derived mesenchymal stem cells (hUCMSCs), which are easily accessible and more proliferative than Schwann cells, were used to prepare an extracellular matrix. We identiifed the morphology and function of hUCMSCs and investi-gated their effect on peripheral nerve regeneration. Compared with a non-coated dish tissue culture, the hUCMSC-derived extracellular matrix enhanced Schwann cell proliferation, upregulated gene and protein expression levels of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor in Schwann cells, and enhanced neurite outgrowth from dorsal root ganglion neurons. These ifndings suggest that the hUCMSC-derived extracellular matrix promotes peripheral nerve repair and can be used as a basis for the rational design of engineered neural niches.     

Neurotrophic factors in the pathogenesis of Rett syndrome

Rett syndrome is characterized by disruption of a period of vigorous brain growth with synapse development. Neurotrophic factors are important regulators of neuronal growth, differentiation, and survival during early brain development. The aims of this study were to study the role of neurotrophic factors in Rett syndrome, specifically whether Rett syndrome has abnormal levels of specific neurotrophic factors in serum and cerebrospinal fluid and whether the changes differ from other neuropediatric patients, for example, those with infantile autism. Four neurotrophic factors were measured: nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1 from the frozen cerebrospinal fluid and from serum (except glial cell line-derived neurotrophic factor) by enzyme-linked immunosorbent assay and cerebrospinal fluid glutamate and aspartate by high-performance liquid chromatography (HPLC) method in patients with Rett syndrome. Insulin-like growth factor 1 was measured from the cerebrospinal fluid of patients with infantile autism. We found low concentrations of cerebrospinal fluid nerve growth factor in patients with Rett syndrome compared with control patients. The serum levels and other cerebrospinal fluid neurotrophic factor levels of the patients did not differ from the controls. Patients with Rett syndrome had high cerebrospinal fluid glutamate levels. Patients with infantile autism had low cerebrospinal fluid insulin-like growth factor 1 levels. Nerve growth factor acts especially on cholinergic neurons of the basal forebrain, whereas insulin-like growth factor 1 acts on cerebellar neurons. In Rett syndrome, the forebrain is more severely affected than the other cortical areas. In autism, many studies show hippocampal or cerebellar pathology. Our findings are in agreement with the different morphologic and neurochemical findings (brain growth, affected brain areas, neurotransmitter metabolism) in the two syndromes. Impairment in dendritic development in Rett syndrome could be the consequence of cholinergic deficiency and of neurotrophic factor/glutamate imbalance. Cholinergic gene expression might be influenced by the Rett syndrome gene directly or via the neurotrophic factor system.     

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves,and muscle re-innervation after peripheral nerve injuries

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large(≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Alth...     

Human umbilical cord blood-derived stem cells and brain-derived neurotrophic factor protect injured optic nerve：viscoelasticity characterization

The optic nerve is a viscoelastic solid-like biomaterial.Its normal stress relaxation and creep properties enable the nerve to resist constant strain and protect it from injury.We hypothesized that stress relaxation and creep properties of the optic nerve change after injury.Moreover,human brain-derived neurotrophic factor or umbilical cord blood-derived stem cells may restore these changes to normal.To validate this hypothesis,a rabbit model of optic nerve injury was established using a clamp approach.At 7 days after injury,the vitreous body received a one-time injection of 50 μg human brain-derived neurotrophic factor or 1 × 106 human umbilical cord blood-derived stem cells.At 30 days after injury,stress relaxation and creep properties of the optic nerve that received treatment had recovered greatly,with pathological changes in the injured optic nerve also noticeably improved.These results suggest that human brain-derived neurotrophic factor or umbilical cord blood-derived stem cell intervention promotes viscoelasticity recovery of injured optic nerves,and thereby contributes to nerve recovery.     

Neurotrophin-4 and glial cell line-derived neurotrophic factor in cerebrospinal fluid from meningitis/encephalitis patients

The neurotrophin-4 and glial cell line-derived neurotrophic factor levels were measured in cerebrospinal fluid from 61 patients with bacterial meningitis, viral meningitis, or encephalitis, and other diseases by means of two-site enzyme-linked immunoassay. Elevated cerebrospinal fluid levels of neurotrophin-4 were demonstrated in four of the 11 patients with bacterial meningitis, and seven of the 23 patients with viral meningitis or encephalitis. None of the other patients demonstrated elevation of the neurotrophin-4 level in cerebrospinal fluid. The neurotrophin-4 levels in cerebrospinal fluid were correlated with the numbers of total and mononuclear cells in patients with viral meningitis/encephalitis. In patients with bacterial meningitis, three of the four patients with elevated neurotrophin-4 levels exhibited persistent abnormalities on computed tomography, and one revealed transient subdural effusion. On the other hand, none of the seven patients without neurotrophin-4 elevation had persistent computed tomography abnormalities, and five patients demonstrated transient computed tomography abnormalities. The glial cell line-derived neurotrophic factor levels were below the detection limit, or only slightly higher than the detection limit, in the patients with or without central nervous system infections. Although the precise roles of neurotrophin-4 and glial cell line-derived neurotrophic factor in central nervous system infections remain to be determined, neurotrophin-4 might play a neuroprotective or immunomodulatory role in central nervous system infections.     

Media that support the growth and differentiation of oligodendrocytes do not induce olfactory ensheathing cells to express a myelinating phenotype

The glial cells that ensheath olfactory axons are referred to as ensheathing cells. In vivo, these non-myelinating glial cells express a mixture of astrocyte-specific and Schwann cell-specific phenotypic features with the former cellular phenotype predominating, but in vitro can assemble a myelin sheath when co-cultured with dorsal root ganglion neurons. Thus, certain in vitro conditions induce ensheathing cells to express a phenotype more like that of a myelinating Schwann cell. The present study addresses whether ensheathing cells will express a myelinating phenotype in neuron-free cultures when fed for 1 to 5 weeks with media shown to promote the growth and differentiation of oligodendrocyte progenitor cells. The ensheathing cell cultures were initiated using the nerve fiber layers (NFL) of rat olfactory bulb primordia. Oligodendrocyte cultures were established from newborn rat neopallium and from the tissue that remained after removing the NFL from the developing olfactory bulb (i.e., the OB-NFL). The cultures were double-labelled with rabbit polyclonal antibodies to S100 or glial fibrillary acidic protein in combination. With the mouse monoclonal antibodies 04, BRD1 [anti-galactocerebroside (anti-GAL-C)], and anti-myelin basic protein (MBP). In some experiments the ensheathing cells were labelled with PKH26 prior to being co-cultured with oligodendrocytes of the OB-NFL. None of the media induced ensheathing cells to express either GAL-C or MBP. However, when 0.5 mM dibutyrylcyclic-AMP (dBcAMP) was added to the medium, ensheathing cells became GAL-C + ve, but remained MBP-ve. The molecular mechanisms that regulate the expression of a myelinating phenotype by ensheathing cells appear to be different from those that operate in oligodendrocytes. © 1994 Wiley-Liss, Inc.     

Hydrophobic dipeptide Leu-Ile protects against neuronal death by inducing brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor synthesis

We investigated whether certain hydrophobic dipeptides, Leu-Ile, Leu-Pro, and Pro-Ile, which partially resemble the site on FK506 that binds to immunophilin, could stimulate glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) synthesis in cultured neurons and found only Leu-Ile to be an active dipeptide. Leu-Ile protected against the death of mesencephalic neurons from wild-type mice but not from mice lacking the BDNF or GDNF gene. Next, we examined the effects of i.p. or i.c.v. administration of Leu-Ile on BDNF and GDNF contents. Both types of administration increased the contents of BDNF and GDNF in the striatum of mice. Also, peripheral administration of Leu-Ile inhibited dopaminergic (DA) denervation caused by unilateral injection of 6-hydroxydopamine (6-OHDA) into the striatum of mice. The number of rotations following a methamphetamine challenge was lower in the Leu-Ile-treated group than in the nontreated group. Next, we compared the calcineurin activity and immunosuppressant activity of Leu-Ile with those of FK506. Leu-Ile was not inhibitory toward calcineurin cellular activity in cultured neuronal cells. Furthermore, Leu-Ile did not suppress concanavalin A (ConA)-induced synthesis/secretion of interleukin-2 by cultured spleen cells, suggesting that the immunosuppressant activity of Leu-Ile may be negligible when used as a therapeutic tool for neurodegenerative diseases.